Coxiella burnetii is a Gram-negative obligate intracellular bacterium that causes acute Q fever and chronic infections in humans. The two principal limitations for Q fever researchers have been 1) lack of genetic tools and 2) the obligate intracellular requirement for cultivation. Recent advances are beginning to resolve these restrictions: a) growth medium using microaerophilic conditions allowing replicate outside of host cells;b) novel techniques for efficient clonal isolation;c) functional Himar1-based transposon mutagenesis system to generate random mutations;and d) transformation with a stable shuttle vector plasmid. The objectives are to generate a mutant library of C. burnetii clones and evaluate phenotypic alteration in vitro and in vivo using C. burnetii, Nine Mile, phase II (RSA439, clone 4;reduced virulence, BL2 containment), as a model for future studies with fully virulent organisms by: 1) developing a transposon mutant clonal library for C. burnetii using a modified isolating clones by micromanipulation, Fluorescent Activated Cell Sorting (FACS) and propagated on agar plates. Each strategy will be optimized and contribute to an ongoing clonal library assembly. Mutants will be characterized genetically for location of insertion using whole genome amplification (WGA), Southern blot, PCR and rescue cloning. 2) Determine the contribution to the pathogenic process in vitro. Clones originating using both media and L929 fibroblast infection systems will be compared with wild type for growth rates in media and in several tissue culture model cells, including tolerance for oxidative stress and survival in primary monocytes/macrophages. 3) Determine the contribution to the pathogenic process for mutants in vivo. Infectivity studies will be performed in SCID mice, which support persistent infection including a moderate disease process with intratracheal RSA439 challenge. A competitive infection model will be established where a pool of mutants will be compared for ability to infect and persistent compared to wild. In vivo imaging using luciferase expression will conveniently characterize replication and dissemination in animals. Growth rates in and histopathologic consequences to lung and spleen will be compared for selected mutations. Complementation of specific mutation using the stable shuttle vector plasmid will confirm that alteration in phenotype is a result of the targeted transposon insertion and not a secondary, undefined mutation, . PUBLIC HEALTH RELEVANCE: It is our expectation that these studies will result in an optimized mutagenesis system for future studies with fully virulent isolates including both acute and chronic pathotypes. We predict that a large collection of mutations will manifest as altered phenotypes and result in identification of genes essential for infectivity, survival in macrophages and colonization and dissemination of organisms in a mouse with defective acquired immunity but functional innate responses. These studies will help establish a fundamental paradigm shift in the molecular pathogenesis studies with this organism. Elucidation of important virulence determinants will provide targets for vaccine design, diagnostic antigens, and therapeutic intervention.